Protein C is a serine protease and naturally occurring anticoagulant that plays a role in the regulation of homeostasis by inactivating Factors V.sub.a and VIII.sub.a in the coagulation cascade. Human protein C is made in vivo primarily in the liver as a single polypeptide of 461 amino acids. This single chain precursor molecule undergoes multiple post-translational modifications including 1) cleavage of a 42 amino acid signal sequence; 2) proteolytic removal from the one chain zymogen of the lysine residue at position 156 and the arginine residue at position 157 to make a 2-chain zymogen form of the molecule, (i.e., a light chain of 155 amino acid residues attached through a disulfide bridge to the serine protease-containing heavy chain of 262 amino acid residues); 3) vitamin K-dependent carboxylation of nine glutamic acid residues clustered in the first 42 amino acids of the light chain, resulting in nine gamma-carboxyglutamic acid residues; and 4) carbohydrate attachment at four sites (one in the light chain and three in the heavy chain). The heavy chain contains the well established serine protease triad of Asp 257, His 211 and Ser 360. Finally, the circulating 2-chain zymogen is activated in vivo by thrombin at a phospholipid surface in the presence of calcium ion. Activation results from removal of a dodecapeptide at the N-terminus of the heavy chain, producing activated protein C (aPC) possessing enzymatic activity.
In addition to the enzymatic activities of aPC within the blood coagulation cascade, aPC also can autodegrade, leading to decreased functionality as an anticoagulant. Applicants have discovered an important degradation pathway. Autodegradation of the N-terminus of the light chain may result in a clip on either side of the histidine residue at position 10. Thus, this degradation pathway yields two inactive products: 1) des(1-9) activated protein C, wherein the first nine N-terminal residues of the light chain have been removed; and 2) des(1-10) activated protein C, wherein the first ten N-terminal residues of the light chain have been removed. This degradation pathway, which has not been previously reported, results in loss of anticoagulant activity due to the removal of the critical GLA residues at positions 6 and 7. Therefore, minimizing the level of the des(1-9) and des(1-10) activated Protein C autodegradation products is important in achieving a potent, high purity, activated protein C pharmaceutical formulation. These variants were previously unknown degradation products and are exceedingly difficult, if not impossible, to remove by conventional purification techniques. Applicants have further discovered that solid-state solubility is significantly enhanced in the presence of a select group of bulking agents.
It is clearly desirable to minimize such degradation of activated protein C in both the solution and lyophilized solid states. Accordingly, these discoveries allow the preparation of potent, high purity, activated protein C formulations which are pharmaceutically elegant to the health care provider.
The present invention provides improved formulations of activated protein C substantially free of such autodegradation products, particularly, des(1-9) and des(1-10) forms of the light chain of activated protein C. Therefore, said formulations are suitable for administration to a patient in need thereof.